John B. March
Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, EH26 0PZ, UK
Introduction and Background to the Problem
Contagious bovine pleuropneumonia (CBPP) is currently the most economically serious disease of cattle in Africa, and in the last decade there has been a substantial re-emergence of the disease, despite vaccination campaigns using freeze dried broth cultures of live attenuated Mycoplasma mycoides subspecies mycoides small colony biotype (Mmm SC) (strain T1 44 or T1 SR). The disease was successfully eradicated from the United Kingdom, North America and much of Western Europe by policy of movement restriction and slaughter during the late 19th century. The disease was also eradicated from Australia by the 1960s, following a successful vaccination campaign that began in the mid-1930s (Newton, 1992). Although other control measures undoubtedly played their part in the successful eradication of CBPP from Australia, the CBPP vaccine, using live broth cultures of Mmm SC strain V5 has been hailed as ‘one of the most important developments in the fight against the disease’ (Newton and Norris, 2000). However, such ‘non-vaccine based’ control measures cannot realistically be applied in Africa due to economic, cultural and social conditions, meaning that effective vaccination is the only realistic policy for CBPP eradication.
Several publications and expert group meetings have reported on the inability of current vaccines to control the disease in Africa (Anon, 1999, 2001; Masiga and Domenech, 1995; Nicholas et al., 2000), and research designed to produce ‘next generation’ vaccines (e.g. ISCOM, capsular polysaccharide conjugate) has proceeded, although with little apparent success (Absurga et al., 1997; Waite and March, 2002). It is likely that further research findings based upon novel vaccine technologies will also be presented at this meeting. My own research group have also been involved in testing newer technologies including recombinant proteins, DNA vaccines, and even derivatives of wheat carbohydrates have been tested by us (the use of flour as a vaccine for CBPP remains a distant goal!). Unfortunately, for all the scientific interest of these technologies, they are unlikely to produce a usable CBPP vaccine for the foreseeable future. Major issues include:
(i) Cost: The financial burden of developing and testing ‘next generation’ vaccines is likely to be in the order of several million dollars at the very minimum, even if much of the animal testing can be carried out in Africa, and to a lower standard of safety than otherwise may be demanded for equivalent ‘Western’ animal vaccines. If equivalent standards of efficacy, safety and licensing were to be demanded then the cost is likely to run into tens if not hundreds of millions of dollars. It is unclear where such funding would come from; the private sector is unlikely to put forward such large sums of money, particularly with little or no possibility of return on investment, and funding by aid agencies and research trusts is few and far between and certainly not of the order of magnitude required.
(ii) Time: Even should sufficient funding be in place, it is likely to take a minimum of 10 years before any new vaccine could be tested, validated and put into production and be ready for widespread use in the field. The intermittent nature of much of today’s research and development funding means that the time taken to produce a vaccine may be even longer. What vaccines are to be used in the meantime?
(iii) Efficacy: Although it is assumed that a ‘next generation’ vaccine against CBPP will prove more efficacious, this is by no means assured. Any such vaccines tested to date have proved to be noticeably unsuccessful (Absurga et al., 1997; Waite and March, 2002), and in some instances have even exacerbated symptoms (Hubschle et al., 2003). Since the basic mechanism(s) of CBPP immunopathology are not yet understood, and since no ‘protective’ antigens (with the possible exception of the capsular polysaccharide) have been specifically identified, the wait for an effective ‘new’ CBPP vaccine may be lengthy.
(iv) Production issues: Current CBPP vaccines are very ‘low tech’ and in fact are exclusively made on location in Africa, with relatively simple equipment, facilities and staff training requirements. It is unlikely that a subunit, recombinant protein or nucleic acid vaccine could be made in situ in Africa without a large investment in infrastructure, training and equipment. For example, it cost roughly US $300,000 to produce sufficient GMP-quality DNA for a malaria vaccine trial currently underway in Africa involving only 2000 subjects (Adrian Hill, personal communication). This is quite clearly a totally uneconomic proposition. If ‘next generation’ vaccines have to be made in the West and imported into Africa this would remove a vital level of autonomy and decision making in the whole process. In addition, where will the funding come from to pay for this infrastructure build or alternatively pay for vaccine importation if it cannot be made locally?
(v) Politics: The control policies involved in a pan-continental disease issues are highly complex, involving both national, international and donor country issues. Agreement and implementation of even the simplest of measures is often extremely difficult to achieve. The complex financial, regulatory, and scientific agendas involved in the development of any new vaccine technology are likely to severely hamper the widespread adoption of the vaccine(s), even assuming that issues (i-iv) above can be adequately addressed.
In isolation these difficulties are formidable; when seen as a whole they appear to be almost insurmountable if it is to be hoped that a new and effective CBPP vaccine will be in place during the next decade. Whilst recent initiatives such as the Wellcome Trust’s Tropical Animal Health initiative, or charities such as the Gates Foundation may be approachable, the timescales involved before we even begin to understand the basic mechanisms of immunopathology, let alone develop ‘next generation’ vaccines means the we must look to what is available now. It is my contention that it is more important to make the best use of current technologies to try and control CBPP today rather that concentrate upon the development of technologies that may not prove effective for at least a generation, if at all. This is all the more important since it is clear that current technologies can be effective if properly used. It could be suggested that current research priorities have more to do with the career requirements of Western scientists than the economic and veterinary needs of the developing world.
It is interesting to note that approximately 10 years have passed since the re-emergence of interest in CBPP following outbreaks in Europe and Africa. Despite several million euros spent in research projects, vaccine trials, workshops, expert group meetings and international scientific meetings, little or no meaningful progress has been made with regard to developing new vaccines and other control measures during this period. One is tempted to ask; could all this money and resource have been better spent? How many vaccination campaigns or serological surveys could this have funded?
Currently used CBPP Vaccines
Almost without exception, all effective CBPP vaccines have been based upon live versions of the disease-causing mycoplasma, either attenuated or not. Current vaccine strains (T1 44 and T1 SR) for CBPP are made from freeze dried broth cultures of live attenuated (Mmm SC) and are generally considered to exhibit poor efficacy and stability (Rweyemamau et al., 1995; Thiaucourt et al., 2000). However, the poor efficacy of vaccines against CBPP appears to be a relatively recent phenomenon, with many reports existing in the literature of successful vaccines based upon live cultures of Mmm SC. The first published account of a successful CBPP vaccine appeared as long ago as 1852, although the procedure (involving the implantation under the skin of the forehead of tiny pieces of diseased tissue from a beast that had succumbed to CBPP) had apparently been in use for some time prior to this. By 1926 vaccination and control of this disease was apparently well under control in parts of Africa. To quote from J. Walker, the Chief Veterinary Research Officer in Kenya, “The serum diagnosis of pleuro-pneumonia.... and preparation of pleuro-pneumonia vaccine are now daily technical routine work’ (Walker, 1929).
CBPP was successfully eradicated from Australia using the V5 broth vaccine, with no real problems regarding either efficacy or thermostability under field conditions every bit as hostile as those likely to be encountered in Africa (Newton and Norris, 2000). As long as the liquid vaccine was kept wrapped in a damp cloth and protected from direct sunlight this was considered adequate protection (Hudson, 1968). Indeed, even vaccine strain KH3J (long regarded as being of negligible protective efficacy in Africa) was successfully used in Australia during the early 1960s (Hudson, 1065).
How can these apparent contradictions in reported vaccine efficacy and stability be reconciled? The recent experiences with the T1 44 vaccine in Namibia showed that it was actually highly effective in bringing CBPP under control: reported disease incidence was reduced from 2794 in 1997 to only 87 in 1999 (Bamhare, 2001). Thus the current vaccine was highly effective when administered as part of a well conducted vaccination campaign, in which (i) high levels of coverage were achieved and, (ii) in which the vaccine was used as quickly as possible following reconstitution (before a significant loss in titre occurred). If these conditions could be achieved over the entire continent, CBPP would be a disease of the past. Unfortunately, it is probably wishful thinking to hope that such well-run vaccination campaigns can take place over much of sub-saharan Africa, but based upon recent research findings there are several recommendations that can be made which should have a significant impact upon the efficacy of the current CBPP vaccines and which can be implemented immediately. What are the main issues currently affecting vaccine efficacy?
Recommendations for Change
Effective buffering of growth media: Assuming that the minimum protective dose of MmmSC is 107 live mycoplasmas per dose (Gilbert and Windsor, 1971), it obviously becomes vitally important to maintain vaccine titre when in the field. Since it is seems clear that current vaccines can provide effective protection when given correctly, and since the emergence of new ‘vaccine resistant’ strains has not been reported (March et al., 1999), the major factor behind poor vaccine efficacy is likely to be sub-optimum bacterial titres. It is known that many vaccine production laboratories do not reach the OIE recommendation of delivering a vaccine at 108 viable mycoplasmas per animal dose (which allows for losses during lyophilisation, storage and transport (Litamoi and Seck, 1999; Rweyemamu et al., 1995). Why is it difficult to achieve and maintain effective titres? A reduction in vaccine pH during culture growth is the most likely explanation. Apart from excessive heat (greater than 43°C), the pH of the growth medium is the principal factor which affects mycoplasma viability (Garba, 1980; Gourlay and MacLeod, 1966; Miles, 1983; Rodwell and Mitchell, 1979; Windsor, 1978). Current vaccine media (e.g. Gourlay’s (Gourlay 1964) and F66 (Provost et al., 1970) are poorly buffered, containing a dibasic (Na2HPO4) phosphate salt only, and exhibit a sharp drop in pH during MmmSC growth. This is mirrored by a rapid reduction in culture viability once the pH begins to fall. In contrast, the growth medium used to produce the successful V5 broth vaccine did not contain glucose (a reducing sugar) and as a result, while the final mycoplasmal titre may have been slightly reduced compared to media containing glucose, the pH did not fall below neutrality (i.e. pH 7.0) (Turner et al., 1935). The result was that vaccines were highly stable for relatively long periods at high ambient temperatures (at least 1 month at 37oC) (Hudson 1968; Turner et al., 1935). If glucose is to be kept in the growth medium, then a buffer system based upon N-[2-hydroxyethyl] piperazine-N’-[2-ethanesulfonic acid] (HEPES) can be used (Waite and March, 2002). This exhibits a 10-fold increase in final titre and markedly increased culture survival compared to contemporary media due to maintenance of a neutral pH during the growth and stationary phases. Yields in excess of 1011 cfu /ml can easily be achieved. Whilst MmmSC cultures in conventional media drop in titre from 1010 /ml to completely inactivated within 2 days at 37°C, the simple addition of HEPES buffer means that the titre at 37°C is still above 108 /ml after 1 month at 37°C, and is 104 /ml after 4 months. An impressive result for such a simple and inexpensive action, with no other changes to current procedures required. The benefit to the user - the vaccine is far more stable. The benefit to the producer - the optimum time for vaccine harvesting is increased from a few hours to several days and the yield is increased 10-fold.
Inclusion of pH indicators: Although high ambient temperatures can evidently lead to MmmSC cell death, cultures will remain stable at 37°C for several weeks as long as a neutral pH is maintained (Turner et al., 1935), and the culture is protected from direct sunlight (Hudson, 1968). It is not until the temperature is above 42°C before rapid cell death occurs. Therefore as long as the culture pH is maintained above roughly pH 6, vaccines should be stable for several days in the field (i.e. without a fall in titre). An extremely simple and inexpensive way to monitor this is to incorporate a simple pH indicator, such as phenol red to the culture medium and /or reconstitution fluid. With a printed pH indicator label attached to the vial, this would provide a rapid and inexpensive visual confirmation of vaccine pH following reconstitution, providing a degree of reassurance and control not currently available to users of CBPP vaccines. A simple visual cut off below which a vaccine should not be used could be incorporated on the label. This simple action would provide a direct benefit to the user.
Similarly, the incorporation of pH indicators should also provide a direct benefit for vaccine producers. Vaccine production should be easier, since a ‘real time’ check on acidity can be made, without the need for any external sampling and pH monitoring with its associated contamination risks. Again, the cost of incorporating such changes would be minimal - no additional equipment or procedures would be required. To encourage the use of pH indicators, FAO/OIE ‘Approved Status’ should only be granted to vaccines including such indicators.
Changes in vaccine reconstitution procedure: The current OIE recommended reconstitution procedure for CBPP vaccines (T1 44 and T1 SR) is to use a solution of 1 molar MgSO4 (Anon, 2001), which has been reported to increase thermostability (Provost, 1970; Provost et al., 1987). However, this procedure was only ever reported for vaccine strain KH3J (not in use today) and has apparently never been tested for strains T1 44 and T1 SR (current vaccine strains). Unfortunately, these strains result in a much more acidic culture during growth than strain KH3J (Gourlay and MacLeod, 1966). This is highly significant, since re-suspension of freeze dried CBPP vaccines in 1M MgSO4 makes the vaccine even more acidic due to precipitation of the phosphate buffer component (present in all current vaccine media) (March et al., 2002). The Mg2+ combines with the phosphate to produce insoluble magnesium orthophosphate, not only removing the buffer component, but releasing free H+ in the process! It is no wonder the vaccines become so acidic. The acidic pH rapidly leads to mycoplasma death and vaccine inactivation; which will also explain why current vaccines are so unstable when reconstituted in the field following the OIE-recommended procedure. Not only does the MgSO4 solution cause a drop in pH, but the culture pH is lower to begin with due to the presence of the reducing sugar glucose and the use of the highly acidifying strains T1 44 and T1 SR. The cumulative effect of all of these changes is a large reduction in pH and stability for ‘modern’ vaccines when compared to pre-1970 vaccine formulations: (i) Compared to strain KH3J, strain T1 44 causes an additional drop of 0.6 pH units during growth. (ii) The addition of glucose compared to its absence in identical media results in a further drop of 0.6 pH units. (iii) Reconstitution using 1M MgSO4 causes an additional drop of up to 2.2 pH units. Current reconstituted vaccines can easily be under pH 5! It is hardly surprising that their stability is so poor compared to the Australian vaccine formulations of a generation ago.
In contrast, when a vaccine culture is reconstituted in buffered saline or water it remains stable for many days at 37°C. In 1M MgSO4 the titre drops by 6 log10 over an 8 hour period. We have observed however, that this effect on culture pH can be largely removed if HEPESbuffered vaccine media (as suggested above) are used March et al., 2002).
Use of phosphate-buffered saline (PBS) as a reconstitution solution: An alternative or additional suggestion to the use of HEPES-buffered growth medium is to use PBS as a reconstitution fluid, in place of 1 M MgSO4. While purified water has the advantage that it does not cause a drop in pH following vaccine reconstitution, it cannot provide any additional buffering capacity of its own. PBS would be useful to restore to neutrality the pH of a borderline vaccine batch (for example around about pH 6 at the time of harvesting). The use of PBS at pH 7.5, containing phenol red as a visual indicator to confirm satisfactory pH prior to injection is the best procedure to maintain vaccine pH and titre following reconstitution (March et al., 2002). This is less important if the vaccine medium already contains HEPES, since it will be properly buffered anyway. If it does not, and is therefore inadequately buffered, the use of PBS as a reconstitution fluid should be mandatory I would suggest.
A range of factors can be seen to affect the final pH of reconstituted T1 44 vaccines, and it would seem that too many variables are present in current methodology to give consistency, particularly since other factors in the field will also affect final vaccine titre (for example, variable ambient temperature, operator skill, length of time reconstituted vaccine is left prior to use). For optimum vaccine efficacy it is important to minimise these sources of variation. Alterations in methodology in order to maintain a neutral vaccine pH following reconstitution should increase vaccine longevity and thus minimise the effect of these other sources of variation.
Conclusions and Recommendations
It is vitally important to maintain vaccine pH near to neutrality to prevent premature spoiling, and unfortunately, the procedures and reagents currently in use and recommended by the OIE do not achieve this. On the basis of findings discussed in this paper, the following recommendations are made. (i) The use of 1 M MgSO4 as a reconstitution fluid in the field should be terminated forthwith. (ii) Phosphate buffered saline should be used as an alternative reconstitution fluid. This should provide an extra measure of security against the generation of an acidic pH following reconstitution, even if a vaccine was desiccated under slightly acidic conditions. (iii) Ideally, HEPES-buffered growth media should be used to prepare vaccine stocks to ensure the pH never drops below pH 7.0 during the growth cycle. (iv) The FAO and OIE to recommend only the use of vaccines and diluents containing pH indicators such as phenol red. With a printed pH indicator label attached to the vial, this would provide rapid and inexpensive visual confirmation of vaccine pH prior to and following reconstitution, providing a degree of reassurance and control not currently available to users of CBPP vaccines.
Hopefully, as the recent experience of Namibia shows, current vaccines can and will be able to make a significant impact upon CBPP disease control. The adoption of these simple and inexpensive measures should have a significant impact upon a disease that is unfortunately as much of a scourge in Africa today as at any time in the past. With proper implementation, it is plausible to hope that by the time any ‘next generation’ CBPP vaccines do finally appear, the disease may already be under control (or even eradicated?) in Africa. I would like to propose that rather than necessarily concentrating on promoting further research, the FAO/OIE/OAUIBAR should perhaps seek large scale funding from agencies such as the Gates Foundation for a concerted vaccination and diagnostic program to eradicate CBPP from the African continent using the tools and technologies already at its disposal. To quote again from the book ‘Clearing a Continent’, which records the eradication of CBPP from Australia during the years leading up to the 1960s ‘On the world scene, eradication under the difficult circumstances in Australia may well encourage countries where the disease persists to embark on a similar course’ (Newton and Norris, 2000). Hopefully this will be so.
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